Clearing conditions for optimal transparency. Clearing conditions for optimal transparency. A, Comparison of ETC temperature and hydrogel composition on tissue transparency and expansion. There were significant main effects of clearing temperature (F(2,24) = 178.9, p < 0.00001) and hydrogel concentration (F(3,24) = 16.2, p < 0.00001) on transparency Tissue appears clearest with 37/55°C ETC and 3% acrylamide, 3% formaldehyde, and 0.025% hydrogel. Tissue expansion is less in the 37/55°C condition compared with either 37°C or 55°C alone. B, Both 55 and 37/55°C produce tissue that is more transparent than 37°C, regardless of hydrogel composition (ps = 0.0002 and 0.0009, respectively). Transmission was measured on a light table under standardized illumination conditions. A reference measurement was made without a sample and was set to 100%. The sample was then imaged on the light table and the percentage light intensity through the specimen was recorded as a percentage of the reference value. C, Brains cleared with the combined 37/55°C clearing protocol expand more in lower concentration hydrogel (3% formaldehyde:3% acrylamide:0.025% bis-acrylamide compared with higher concentration hydrogel (4% formaldehyde:4% acrylamide:0.05% bis-acrylamide). The conditions for producing an optimal combination of stable and clear tissue is to polymerize the tissue with hydrogel composed of 4% acrylamide, 4% formaldehyde, and 0.05% bis-acrylamide and then clear the tissue with 5 d of ETC (4 d at 37°C and 1 d at 55°C). Jonathan R. Epp et al. eneuro 2015;2:ENEURO.0022-15.2015 ©2015 by Society for Neuroscience